The effect that BglI and PstI have on a plasmid
In Biotech 1015 we learned how to purify DNA and extract plasmids to help us identify
fragmentation. A plasmid is a segment of DNA used to transfer genetic material from one cell to
another. Plasmids are used for inserting other strands of DNA into a plasmid. Restriction
enzymes are a group of enzymes that catalyze to the cleavage of DNA plasmids at specific sites.
Restriction enzymes help us find fragmentation of plasmids. Gel electrophoresis is the
predominant way to look at DNA fragments and it is easily comparable to other gels. For a DNA
gel first you need to make an agarose gel then you will load the DNA into the gel and run for a
period of time. Once it has run for a certain period of time you will take it out and read the gel
under a UV light. Our goal for this experiment was to see if we could identify a plasmid by
running it through tests that we have learned through out this semester. The goal of this project is
important because it shows what we have learned in lab. I was given a plasmid that I will cut
with enzymes of my choice and run in a gel to see if I am able to identify my plasmid.
Methods
My code name for my plasmid was Actinium (Ac). To determine the concentration of the
plasmid you can use Beer Lambert’s law and a Spectrophotometer. Our Ladder came from NEB
labs (New England Biolabs). The enzymes and buffer that I used were BglI, PstI and NEBuffer
3. All the enzymes and buffers came from NEB labs. For my single digest I used PstI and for my
double digest I used PstI and BglI. I started out by incubating my PCR tubes with plasmid and
enzymes at 37o C for 9 hours. While those incubated I made a 1% Agarose gel: 1.Measure out
0.5 grams of Agarose 2. Add 50mL of 1X TAE 3. Microwave for 30 seconds then add 15
seconds until liquid is clear. 4. Cool liquid in ice. 5. Pour in gel tray with a 10 well comb and let
sit until firm. I used a 1X TAE buffer solution to run my gel. I ran my gel at 150 volts for 30
minutes. Once the gel ran for 30 minutes I used a UV Transilluminator to look and print a picture
of my gel. After I was done with my gel I compared the picture of my gel with fragmentations
from NEBcutter. For this project the main website I used to look up fragmentations was
NEBcutter.
Results and Conclusion
Our starting concentration of plasmid is 17.4 ug/mL
6
3.3
10
8
3.4
6
2.6
5
1.5
4
3
2
1.5
1
0.5
3.3
Ladder
10.0
8.0
6.0
5.0
4.0
3.0
2.0
1.5
1.0
0.5
Uncut
3.3
PstI
BglI & PstI
6.0
3.4
3.3
2.6
1.5
NEBcutter fragment tables
pAMP single
pBLU single
pGEM single (cannot be cut by
PstI)
pGLO single
pAMP Double
pGEM Double (PstI does not cut)
pBLU Double
pGEM Double
NEBcutter restriction maps
pAMP single
pBLU single
pGEM single (PstI does not digest)
pGLO single
pAMP Double
pGEM Double (PstI does not digest)
pBLU Double
pGLO Double
Overall I was successful on identifying my plasmid. I identify it as pGLO. If I had more
time for this project I would run the test again and check my results. I also might pick an enzyme
that would digest every plasmid instead of only some of the plasmids. Next time I will incubate
my PCR tubes for more than 9 hours and see how incubation time affects the plasmid results.
References
o "DNA Restriction" Biology Animation Library: DNA Learning Center. (n.d.). DNA
Learning Center. Retrieved December 8, 2011, from
http://www.dnalc.org/resources/animations/restriction.html
o Double Digest Finder, NEB. (n.d.). Reagents for the Life Science Industry | New England
Biolabs. Retrieved December 8, 2011, from
http://www.neb.com/nebecomm/DoubleDigestCalculator.asp?
o NEBcutter V2.0. (n.d.). tools.neb.com. Retrieved December 8, 2011, from
http://tools.neb.com/NEBcutter2/
o Plasmid. (n.d.). Dictionary.com. Retrieved December 8, 2011, from
dictionary.reference.com/browse/plasmid
o Reagents for the Life Science Industry | New England Biolabs. (n.d.). Reagents for the
Life Science Industry | New England Biolabs. Retrieved December 8, 2011, from
http://www.neb.com/nebecomm/default.asp
o Restriction Enzyme. (n.d.). Dictionary.com. Retrieved December 7, 2011, from
dictionary.reference.com/browse/restriction+enzyme